Project Summary Invasive infections due to the ?superbug? methicillin-resistant Staphylococcus aureus (MRSA) are responsible for more deaths than any other drug-resistant bacterial pathogen in the USA. The glycopeptide (GP), vanco- mycin, is the standard of care for the treatment of MRSA, but therapeutic failures are common. Vancomycin is closely related to two other classes of antibiotics that are also important for the treatment of MRSA infections, the lipopeptides (LP) and the lipoglycopeptides (LGP). Drugs from each of these classes can select for cross- resistance to the others to various degrees, but the mechanisms behind this cross-resistance are not well un- derstood. In addition, there is a critical need to develop new diagnostics that can predict drug response to GP, LP, and LGP antimicrobials, and to develop novel therapies that can modulate resistance. Recently, using an innovative lipidomic approach we have observed significant and characteristic changes in lipid metabolism as- sociated with resistance to each class of drugs (GP, LP, and LGP). We hypothesize that lipidomic signatures in the cell membrane reflect specific antibiotic resistance mechanisms and that these lipidomic signatures can be modified by other small molecules to favor a susceptible phenotype. We propose to comprehensively inter- rogate the mechanisms of resistance and cross-resistance to GP, LP, and LGP antimicrobials in MRSA by in- tegrated lipidomics, genomics, and transcriptomics. In AIM 1, our strategy will focus on elucidating the role of altered lipid metabolism in the development of resistant phenotypes in in vitro-selected strains that are re- sistant to GP, LP, and/or LGP using comprehensive lipidomics, coupled with genomics, transcriptomics, stand- ard and advanced susceptibility testing, and quantitative biophysical assessment of cell wall and cell mem- brane properties. Contribution of each gene mutation to the resistant phenotypes will be elucidated by similar characterization of single-gene mutants generated by allelic replacement. In AIM 2, we will test the hypothesis that ?-lactams modulate the susceptibility of MRSA to GP, LP, and LGP, at least in part, by modifying the lipid composition of the bacteria. We will examine the synergistic interactions between the study drugs and ?- lactams by the integrated omics approach, which would yield insights into the mechanisms of ?-lactam synergy with these agents and the ?-lactam ?seesaw effect?. In AIM 3, we will evaluate the sensitivity of our lipidomic technique to identify sub-MIC differences in susceptibility and predict response to clinically relevant GP, LP, and LGP exposures. We anticipate that this strategy will yield valuable insight into the role of altered lipid me- tabolism in producing antimicrobial resistance. This work is significant because the pathways identified as crit- ical to resistance and susceptible to modulation will lay the foundation for further studies that could lead to re- sistance prevention therapeutics and advanced diagnostics, which in turn will improve human health. The in- novation of this proposal includes the novel lipidomics methodology, the integrated omics approach, and cor- relation of lipidomic signatures with clinically relevant pharmacodynamics endpoints.